Internal calibrants allow high accuracy peptide matching between MALDI imaging MS and LC-MS/MS

One of the important challenges for MALDI imaging mass spectrometry (MALDI-IMS) is the unambiguous identification of measured analytes. One way to do this is to match tryptic peptide MALDI-IMS m/z values with LC-MS/MS identified m/z values. Matching using current MALDI-TOF/TOF MS instruments is difficult due to the variability of in situ time-of-flight (TOF) m/z measurements. This variability is currently addressed using external calibration, which limits achievable mass accuracy for MALDI-IMS and makes it difficult to match these data to downstream LC-MS/MS results. To overcome this challenge, the work presented here details a method for internally calibrating data sets generated from tryptic peptide MALDI-IMS on formalin-fixed paraffin-embedded sections of ovarian cancer. By calibrating all spectra to internal peak features the m/z error for matches made between MALDI-IMS m/z values and LC-MS/MS identified peptide m/z values was significantly reduced. This improvement was confirmed by follow up matching of LC-MS/MS spectra to in situ MS/MS spectra from the same m/z peak features. The sum of the data presented here indicates that internal calibrants should be a standard component of tryptic peptide MALDI-IMS experiments.     

Difference and consensus of whole cell Salmonella enterica subsp. enterica serovars matrix-assisted laser desorption/ionization time-of-flight mass spectrometry spectra

AIM: Application of MALDI-TOF MS for characterization of strains of Salmonella enterica subsp. enterica. METHODS AND RESULTS: Whole cells were analysed by MALDI-TOF MS. Spectra with a maximum of 500 mass peaks between (m/z) 0 and 25000 were examined for consensus peaks manually and by a computer software algorithm. Consensus peaks were observed by both methods for spectra of Salmonella enterica serovars Derby, Hadar, Virchow, Anatum, Typhimurium and Enteritidis. CONCLUSIONS: Differences in numbers of consensus peaks in spectra obtained by manual and computer comparison indicated that development of the software involving statistical analysis of peak accuracy is necessary. SIGNIFICANCE AND IMPACT OF THE STUDY: Development of an analysis system for peak profiles in whole cell MALDI-TOF MS spectra to enable intra and interlaboratory comparison.     

Identification and validation of a potential lung cancer serum biomarker detected by matrix-assisted laser desorption/ionization-time of flight spectra analysis

Many abnormalities detected in the thorax by routine conventional imaging studies are benign, yet all require further evaluation because of the concern for cancer. To address this deficiency and develop a serum biomarker for lung cancer, we designed a matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) based platform to display the proteins present in the serum of patients with or without lung cancer, and then challenged the scientific community to analyze these data with the aim of determining specific ion signal differences among the resulting spectra. The most statistically significant ion peak identified by the various analysis algorithms that differentiated the serum of patients with lung cancer from the serum of individuals without lung cancer was found at m/z 11,702. We identified the protein responsible for this ion peak as serum amyloid A (SAA; M(r) = 11,682.7) by partial purification followed by in-gel digestion and peptide mapping. By enzyme-linked immunosorbent assay, we showed SAA to be present at 286 ng/mL in the serum of cancer patients vs. 34.1 ng/mL in the serum of individuals without cancer. These data suggest that the combination of MALDI-TOF MS and computer analysis can be a powerful tool in the search for serum biomarkers of lung cancer and other diseases.     

Analysis of MALDI-TOF mass spectrometry data for discovery of peptide and glycan biomarkers of hepatocellular carcinoma

This paper presents computational methods to analyze MALDI-TOF mass spectrometry data for quantitative comparison of peptides and glycans in serum. The methods are applied to identify candidate biomarkers in serum samples of 203 participants from Egypt; 73 hepatocellular carcinoma (HCC) cases, 52 patients with chronic liver disease (CLD) consisting of cirrhosis and fibrosis cases, and 78 population controls. Two complementary sample preparation methods were applied prior to generating mass spectra: (1) low molecular weight (LMW) enrichment of each serum sample was carried out for MALDI-TOF quantification of peptides, and (2) glycans were enzymatically released from proteins in each serum sample and permethylated for MALDI-TOF quantification of glycans. A peak selection algorithm was applied to identify the most useful peptide and glycan peaks for accurate detection of HCC cases from high-risk population of patients with CLD. In addition to global peaks selected by the whole population based approach, where identically labeled patients are treated as a single group, subgroup-specific peaks were identified by searching for peaks that are differentially abundant in a subgroup of patients only. The peak selection process was preceded by peak screening, where we eliminated peaks that have significant association with covariates such as age, gender, and viral infection based on the peptide and glycan spectra from population controls. The performance of the selected peptide and glycan peaks was evaluated in terms of their ability in detecting HCC cases from patients with CLD in a blinded validation set and through the cross-validation method. Finally, we investigated the possibility of using both peptides and glycans in a panel to enhance the diagnostic capability of these candidate markers. Further evaluation is needed to examine the potential clinical utility of the candidate peptide and glycan markers identified in this study.     

Enrichment of low molecular weight fraction of serum for MS analysis of peptides associated with hepatocellular carcinoma

A challenging aspect of biomarker discovery in serum is the interference of abundant proteins with identification of disease-related proteins and peptides. This study describes enrichment of serum by denaturing ultrafiltration, which enables an efficient profiling and identification of peptides up to 5 kDa. We consistently detect several hundred peptide-peaks in MALDI-TOF and SELDI-TOF spectra of enriched serum. The sample preparation is fast and reproducible with an average CV for all 276 peaks in the MALDI-TOF spectrum of 11%. Compared to unenriched serum, the number of peaks in enriched spectra is 4 times higher at an S/N ratio of 5 and 20 times higher at an S/N ratio of 10. To demonstrate utility of the methods, we compared 20 enriched sera of patients with hepatocellular carcinoma (HCC) and 20 age-matched controls using MALDI-TOF. The comparison of 332 peaks at p < 0.001 identified 45 differentially abundant peaks that classified HCC with 90% accuracy in this small pilot study. Direct TOF/TOF sequencing of the most abundant peptide matches with high probability des-Ala-fibrinopeptide A. This study shows that enrichment of the low molecular weight fraction of serum facilitates an efficient discovery of peptides that could serve as biomarkers for detection of HCC as well as other diseases.     

Platelet factor-4 is an indicator of blood count recovery in acute myeloid leukemia patients in complete remission

To investigate whether serum biomarkers can be used to indicate the responsiveness of acute myeloid leukemia to remission induction chemotherapy, we performed MALDI-TOF protein profile analysis of patient sera. The resulting spectra revealed a protein (or peptide) peak at m/z 7764 that varied in intensity; its intensity was much higher in samples from patients in complete remission than in those from patients with resistant disease or in samples taken prior to treatment (at the time of diagnosis). Using fractionation, trypsin digestion, MS/MS, and protein molecular weight analyses, we identified the m/z 7764 protein as platelet factor-4 (PF4). This identification was confirmed by a magnetic bead-based MALDI immunoassay. Statistical comparison of PF4 levels and platelet counts in patient sera revealed a significant positive correlation between the two variables. This study demonstrates that PF4 protein levels are a good indicator for the recovery of blood count in the complete remission of acute myeloid leukemia. The linear positive correlation curve indicates that blood count recovery of platelets to >100,000/mm(3) is equivalent to a serum PF4 recovery level of >2.492 microg/ml.     

Detection of pre-neoplastic and neoplastic prostate disease by MALDI profiling of urine

The heterogeneous progression to the development of prostate cancer (PCa) has precluded effective early detection screens. Existing prostate cancer screening paradigms have relatively poor specificity for cancer relative to other prostate diseases, commonly benign prostatic hyperplasia (BPH). A method for discrimination of BPH, HGPIN, and PCa urine proteome was developed through testing 407 patient samples using matrix assisted laser desorption-mass spectrometry time of flight (MALDI-TOF). Urine samples were adsorbed to reverse phase resin, washed, and the eluant spotted directly for MALDI-TOF analysis of peptides. The processing resolved over 130 verifiable signals of a mass range of 1000-5000 m/z to suggest 71.2% specificity and 67.4% sensitivity in discriminating PCa vs. BPH. Comparing BPH and HGPIN resulted in 73.6% specificity and 69.2% sensitivity. Comparing PCa and HGPIN resulted in 80.8% specificity and 81.0% sensitivity. The high throughput, low-cost assay method developed is amenable for large patient numbers required for supporting biomarker identification.     

Determination of new biomarkers to monitor the dietary consumption of isothiocyanates

Isothiocyanates (ITCs) found in cruciferous vegetables have been associated with a reduced cancer risk in humans. We determined serum albumin adducts of allyl isothiocyanate (AITC), benzylisothiocyanate (BITC), phenylethylisothiocyanate (PEITC) and sulforaphane (SFN) in 85 healthy men from a dietary, randomized, controlled trial. After enzymatic digestion of albumin we determined the adducts of the ITCs with lysine (Lys) using liquid chromatography–tandem mass spectrometry. At the beginning of the study (and after 4 weeks) 4.7% (2.4%), 48.2% (35.3%), 5.9% (10.6%), and 24.7% (23.5%) of the samples were found positive for AITC-Lys, BITC-Lys, PEITC-Lys and SFN-Lys, respectively. This method enables the quantification of ITC adducts in albumin from large, prospective studies on diet and cancer.     

Molecular identification and population dynamics of two species of Pemphigus (Homoptera: Pemphidae) on cabbage

The poplar petiole gall aphid, Pemphigus populitransversus Riley, has been one of the major pests on cruciferous vegetable in the Rio Grande Valley (LRGV) of Texas since the late 1940s. It normally migrates from poplar trees to cruciferous vegetables in the fall, and migrates back to the trees in early spring of the coming year. Some root‐feeding aphids were found on cruciferous vegetables in late spring and early summer in 1998 and the following years. Those aphids have been identified as Pemphigus obesinymphae Moran. This discovery completely changed the current knowledge about the root‐feeding aphids on cruciferous vegetables in the LRGV. Due to their small size, morphological and feeding similarities between P. populitransversus and P. obesinymphae, their identification and distinction are difficult. In this study, random amplification of polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) were used to distinguish these two species over a period of time when the two species occurred together, or separately, in cabbage fields. The two species occurred on cabbage at different times of the year, and overlapped from October to June. From May to October, both species migrated to their primary hosts. The apterous aphids found on cabbage in winter contained mainly P. obesinymphae, whereas in early spring more apterous P. populitransversus were recovered. The root‐feeding aphids would feed on cabbage plants as long as this host was available even during the hot, dry summer in the LRGV, although their populations were generally low. Both RAPD and AFLP techniques were efficient in discriminating the two species that showed obviously genetic variability. These molecular techniques confirmed the existence of the two aphid species in apterous samples collected from the soil in cabbage fields in the LRGV, and the results performed by RAPD were confirmed by AFLP. Furthermore, the results suggest that RAPD technique was a better choice despite its reproducibility problem, as it was less time‐consuming and required less technology, labor and expense than AFLP.     

Spectroscopic detection and identification of infected cells with herpes viruses

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and Fourier transform infrared (FTIR) microspectroscopy were previously applied for the identification of various biological samples. In the present study, normal cells in culture and cells infected with herpes simplex virus type 2 (HSV-2) or varicella-zoster virus (VZV) were analyzed by MALDI-TOF and FTIR microscopy. Specific spectral biomarkers for rapid and reliable monitoring and identification of infected cells and probably for the discrimination between these viruses were searched. The results show consistent spectral peaks in all examined normal uninfected human fibroblast cells both in MALDI-T0F and FTIR microscopy. In HSV-2- or VZV-infected cells, two unique peaks for each appeared at m/z 5397 and 5813 or at m/z 3501 and 4951, respectively, in MALDI-TOF spectra. In addition, several peaks that appeared in control uninfected cells at the region m/z 13,000-20,000 disappeared completely in all examined infected samples. When these infected cells were examined by FTIR microscopy, a band at 859 cm(-1) in control uninfected cells was significantly shifted to 854 cm(-1) in both HSV2- and VZV-infected cells. In addition, phosphate levels were considerably increased in all infected cells compared to normal uninfected cells. These parameters could be used as a basis for developing a spectral method for the detection and identification of cells infected with herpes viruses.     

Discrimination of intact mycobacteria at the strain level: a combined MALDI-TOF MS and biostatistical analysis

New methodologies for surveillance and identification of Mycobacterium tuberculosis are required to stem the spread of disease worldwide. In addition, the ability to discriminate mycobacteria at the strain level may be important to contact or source case investigations. To this end, we are developing MALDI-TOF MS methods for the identification of M. tuberculosis in culture. In this report, we describe the application of MALDI-TOF MS, as well as statistical analysis including linear discriminant and random forest analysis, to 16 medically relevant strains from four species of mycobacteria, M. tuberculosis, M. avium, M. intracellulare, and M. kansasii. Although species discrimination can be accomplished on the basis of unique m/z values observed in the MS fingerprint spectrum, discrimination at the strain level is predicted on the relative abundance of shared m/z values among strains within a species. For the 16 mycobacterial strains investigated in the present study, it is possible to unambiguously identify strains within a species on the basis of MALDI-TOF MS data. The error rate for classification of individual strains using linear discriminant analysis was 0.053 using 37 m/z variables, whereas the error rate for classification of individual strains using random forest analysis was 0.023 using only 18 m/z variables. In addition, using random forest analysis of MALDI-TOF MS data, it was possible to correctly classify bacterial strains as either M. tuberculosis or non-tuberculous with 100% accuracy.     

Peptidome profiling of induced sputum by mesoporous silica beads and MALDI-TOF MS for non-invasive biomarker discovery of chronic inflammatory lung diseases

Induced sputum is recognized as being of increasing importance for the diagnosis and monitoring of chronic inflammatory lung diseases. The main purpose of this study is to provide a valid approach to better fractionate and characterize the still under-estimated low-molecular weight proteome of induced sputum by using mesoporous silica beads (MSBs) SPE coupled to MALDI-TOF MS. Sputum peptides were captured from both derivatized and non-derivatized MSBs and then profiled by MALDI-TOF MS. Depending on the chemical groups present on the mesoporous surface, complex peptide mixtures were extracted from induced sputum and converted into reproducible MALDI profiles. The number of peaks detected as a function of S/N was evaluated for each mesoporous surface. More than 400 peaks with an S/N>5 were obtained in comparison to 200 peaks detected without MSBs. Additionally, as a proof-of-principle, we investigated the ability of this platform to discriminate between the "sputome" of patients with asthma and chronic obstructive pulmonary disease, and between these groups and those of healthy control subjects. Six m/z peaks emerged as potential diagnostic peptidic patterns able to differentiate these inflammatory airway diseases in the sputome range. Human α-defensins (human neutrophil peptide (HNP)1, HNP2, HNP3) and three C-terminal amidated peptides, one of which is phosphorylated on serine, were identified by MALDI-TOF/TOF MS. These findings may contribute to defining a high-throughput screening MS-based platform for monitoring key peptidic-biomarkers for inflammatory and chronic respiratory diseases in induced sputum samples.     

Analysis of Human Proteome Organization Plasma Proteome Project (HUPO PPP) reference specimens using surface enhanced laser desorption/ionization-time of flight (SELDI-TOF) mass spectrometry: multi-institution correlation of spectra and identification of biomarkers

We report on a multicenter analysis of HUPO reference specimens using SELDI-TOF MS. Eight sites submitted data obtained from serum and plasma reference specimen analysis. Spectra from five sites passed preliminary quality assurance tests and were subjected to further analysis. Intralaboratory CVs varied from 15 to 43%. A correlation coefficient matrix generated using data from these five sites demonstrated high level of correlation, with values >0.7 on 37 of 42 spectra. More than 50 peaks were differentially present among the various sample types, as observed on three chip surfaces. Additionally, peaks at approximately 9200 and approximately 15,950 m/z were present only in select reference specimens. Chromatographic fractionation using anion-exchange, membrane cutoff, and reverse phase chromatography, was employed for protein purification of the approximately 9200 m/z peak. It was identified as the haptoglobin alpha subunit after peptide mass fingerprinting and high-resolution MS/MS analysis. The differential expression of this protein was confirmed by Western blot analysis. These pilot studies demonstrate the potential of the SELDI platform for reproducible and consistent analysis of serum/plasma across multiple sites and also for targeted biomarker discovery and protein identification. This approach could be exploited for population-based studies in all phases of the HUPO PPP.     

Rapid Characterization of Microalgae and Microalgae Mixtures Using Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS)

Current molecular methods to characterize microalgae are time-intensive and expensive. Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) may represent a rapid and economical alternative approach. The objectives of this study were to determine whether MALDI-TOF MS can be used to: 1) differentiate microalgae at the species and strain levels and 2) characterize simple microalgal mixtures. A common protein extraction sample preparation method was used to facilitate rapid mass spectrometry-based analysis of 31 microalgae. Each yielded spectra containing between 6 and 56 peaks in the m/z 2,000 to 20,000 range. The taxonomic resolution of this approach appeared higher than that of 18S rDNA sequence analysis. For example, two strains of Scenedesmus acutus differed only by two 18S rDNA nucleotides, but yielded distinct MALDI-TOF mass spectra. Mixtures of two and three microalgae yielded relatively complex spectra that contained peaks associated with members of each mixture. Interestingly, though, mixture-specific peaks were observed at m/z 11,048 and 11,230. Our results suggest that MALDI-TOF MS affords rapid characterization of individual microalgae and simple microalgal mixtures.     

Highly efficient classification and identification of human pathogenic bacteria by MALDI-TOF MS

Accurate and rapid identification of pathogenic microorganisms is of critical importance in disease treatment and public health. Conventional work flows are time-consuming, and procedures are multifaceted. MS can be an alternative but is limited by low efficiency for amino acid sequencing as well as low reproducibility for spectrum fingerprinting. We systematically analyzed the feasibility of applying MS for rapid and accurate bacterial identification. Directly applying bacterial colonies without further protein extraction to MALDI-TOF MS analysis revealed rich peak contents and high reproducibility. The MS spectra derived from 57 isolates comprising six human pathogenic bacterial species were analyzed using both unsupervised hierarchical clustering and supervised model construction via the Genetic Algorithm. Hierarchical clustering analysis categorized the spectra into six groups precisely corresponding to the six bacterial species. Precise classification was also maintained in an independently prepared set of bacteria even when the numbers of m/z values were reduced to six. In parallel, classification models were constructed via Genetic Algorithm analysis. A model containing 18 m/z values accurately classified independently prepared bacteria and identified those species originally not used for model construction. Moreover bacteria fewer than 10(4) cells and different species in bacterial mixtures were identified using the classification model approach. In conclusion, the application of MALDI-TOF MS in combination with a suitable model construction provides a highly accurate method for bacterial classification and identification. The approach can identify bacteria with low abundance even in mixed flora, suggesting that a rapid and accurate bacterial identification using MS techniques even before culture can be attained in the near future.     

Improved candidate biomarker detection based on mass spectrometry data using the Hilbert-Huang transform

Mass spectrometry biomarker discovery may assist patient's diagnosis in time and realize the characteristics of new diseases. Our previous work built a preprocess method called HHTmass which is capable of removing noise, but HHTmass only a proof of principle to be peak detectable and did not tested for peak reappearance rate and used on medical data. We developed a modified version of biomarker discovery method called Enhance HHTMass (E-HHTMass) for MALDI-TOF and SELDI-TOF mass spectrometry data which improved old HHTMass method by removing the interpolation and the biomarker discovery process. E-HHTMass integrates the preprocessing and classification functions to identify significant peaks. The results show that most known biomarker can be found and high peak appearance rate achieved comparing to MSCAP and old HHTMass2. E-HHTMass is able to adapt to spectra with a small increasing interval. In addition, new peaks are detected which can be potential biomarker after further validation.     

A pilot study of salivary N-glycome in HBV-induced chronic hepatitis,cirrhosis, and hepatocellular carcinoma

Hepatitis B is a potentially life-threatening liver infection caused by the hepatitis B virus (HBV), which can lead to chronic liver disease and put people at high risk of death from cirrhosis of the liver and liver cancer. However, little is known about the correlation of salivary N-linked glycans related to HBV-infected liver diseases. Here we investigated N-linked glycome in saliva from 200 subjects (50 healthy volunteers (HV), 40 HBV-infected patients (HB), 50 cirrhosis patients (HC), and 60 hepatocellular carcinoma patients (HCC) using MALDI-TOF/TOF-MS. Representative MS spectra of N-glycans with signal-to-noise ratios >6 were annotated using the GlycoWorkbench program. A total of 40, 47, 29, and 33 N-glycan peaks were identified and annotated from HV, HB, HC, and HCC groups, respectively. There were 15 N-glycan peaks (e.g., m/z 1647.587, 1688.613 and 2101.755) were present in all groups. Three N-glycan peaks (m/z 2596.925, 2756.962, and 2921.031) were unique in HV group, 2 N-glycan peaks (m/z 1898.676 and 1971.692) were unique in HB group, 5 N-glycan peaks (m/z 1954.677, 2507.914, 2580.930, 2637.952, and 3092.120) were unique in HC group, and 3 N-glycan peaks (m/z 2240.830, 2507.914, and 3931.338) were unique in HCC group. The proportion of fucosylated N-glycans was apparently increased in the HCC group (84.8%) than in any other group (73.1% ± 0.01), however, the proportion of sialylated N-glycans was decreased in HCC group (12.1%) than in any other group (17.23% ± 0.003). Our data provide pivotal information to distinguish between HBV-associated hepatitis, cirrhosis and HCC, and facilitate the discovery of biomarkers for HCC during its early stages based on precise alterations of N-linked glycans in saliva.     

Simultaneous determination of urinary androgen glucuronides by high temperature gas chromatography-mass spectrometry with selected ion monitoring

An efficient procedure is described for the simultaneous determination of 9 androgen glucuronides including androsterone, etiocholanolone, 11-ketoandrosterone, 11-ketoetiocholanolone, 11beta-hydroxyandrosterone, 11beta-hydroxyetiocholanolone, and dehydroepiandrosterone (DHEA) in 3-glucuronide form and dihydrotestosterone (DHT) and testosterone in 17-glucuronide form from urine specimens. The method involves solid-phase extraction of the urinary steroids using Serdolit PAD-1 resin, with subsequent conversion to methyl ester-trimethylsilyl (Me-TMS) ether derivatives for the direct analysis by gas chromatography-mass spectrometry (GC-MS) using high temperature MXT-1 (Silcosteel-treated stainless steel) capillary column. Upon split injection of Me-TMS steroids at 330 degrees C into the MXT-1 capillary column initially maintained at 300 degrees C then programmed to 322 degrees C at 2 degrees C/min, each androgen glucuronide was well separated in excellent peak shape. The characteristic ions at m/z 217 constituting the base peaks in the electron-impact (20 eV) mass spectra for most steroids permitted their sensitive detection by GC-MS with selected-ion monitoring (SIM), whereas base peak ion at m/z 271 was used for the SIM of dehydroepiandrosterone-3-glucuronide. The detection limits for SIM of most of the steroids were 15 pg except for the 3-glucuronides of 11-ketoandrosterone and 11-ketoetiocholanolone, which could be detected down to 20 pg. The SIM responses were linear with correlation coefficients varying from 0.981 to 0.993 in the concentration range of 20 to 3000 ng/ml for the androgens studied. When applied to urine samples, the present method allowed rapid screening for the 7 androgens in their glucuro-conjugated forms simultaneously with good overall precision and accuracy within the normal concentration ranges of 15.1 to 3124.6 ng/ml.     

HPLC and GC/MS determination of deuterated vitamin K (phylloquinone) in human serum after ingestion of deuterium-labeled broccoli

The ability to intrinsically label plant constituents with stable isotopes has the potential to advance the study of vitamin K-absorption and metabolism in humans. Broccoli, a primary food source of phylloquinone (VK-1), was grown hydroponically using 31 atom % deuterium oxide in order to label VK-1 within the food matrix. Deuterium-labeled broccoli (115 g; 168 &mgr;g VK-1) was fed to one male subject in a single serving. Multiple serum samples were drawn throughout the subsequent 24-hr period. Reversed-phase HPLC was used to extract and purify VK-1 in both broccoli and serum. Ion abundances of the deuterium-labeled and unlabeled (endogenous) VK-1 were determined using GC/MS in negative chemical ionization mode. No sample derivatization was required. Endogenous VK-1 produced isotopomers from m/z 450 to m/z 453. The labeled VK-1 isotopomers in the broccoli were from m/z 452 to m/z 467, with the most abundant isotopomer being m/z 458 (14.1% of total labeled VK-1). The GC/MS chromatograms from serum revealed both endogenous VK-1 and VK-1 derived from the deuterium-labeled broccoli. The profile of labeled VK-1 isotopomers in serum was identical to the VK-1 isotopomer profile in labeled broccoli, indicating that no deuterium was lost due to exchange either in the body or in sample preparation. At 4 hr following broccoli intake, there was an 81.1% enrichment of phylloquinone in serum; labeled VK-1 was no longer detectable in serum at 24 hr. Use of isotope labeled vegetables enables one to discriminate exogenous intake of VK-1 from endogenous pools and ultimately to determine bioavailability of VK-1 from foods.